Mutations in connexin genes lead to human disease, suggesting that gap junctional communication is required for normal tissue homoeostasis and function. For example, missense mutations in human CX43 cause skeletal malformations and other pleiotropic phenotypes associated with the autosomal dominant oculodentodigital dysplasia (ODDD). It is unclear how missense mutations might cause disease phenotypes. One possibility is that the mutations act in a "dominant-negative" manner, effectively modifying or inhibiting the normal coupling properties of the Cx43 gap junctions. However, it has been difficult to correlate directly the function of particular missense alleles with particular phenotypes using mammalian systems. Mutations in zebrafish cx43 cause the short fin phenotype, characterized by defects in the length of bony fin ray segments and reduced levels of cell proliferation. Using the relatively simple system of zebrafish fin growth, it has been possible to correlate defects in fin length, segment length, and cell proliferation using three missense alleles of zebrafish cx43. The zebrafish genome also contains a cx43-like gene called cx40.8. Interestingly, Cx40.8 is expressed in the same cells as Cx43 during fin regeneration. Unlike Cx43, Cx40.8 appears to localize to the Golgi apparatus rather than primarily at the plasma membrane. Yet, knock-down of Cx40.8 causes similar defects in cell proliferation and segment length as reduced Cx43 function, indicating that Cx40.8 plays an active role in regulating fin growth. One possibility is that Cx40.8 oligomerizes with Cx43 and directly modifies the activity of Cx43 gap junctions. Thus, zebrafish Cx43 and Cx40.8 might represent a natural system to examine Cx43 hetero-oligomerization and its effects on gap junction assembly and coupling properties. Specific Aims 1 and 2 of this application are to determine if Cx40.8 physically interacts with Cx43 in vivo, and if Cx40.8/Cx43 gap junctions exhibit distinct electrical coupling properties. Evidence for this interaction and regulation may reveal an endogenous mechanism that regulates gap junctional coupling. Specific Aim 3 is to identify the molecular mechanism that allows Cx40.8 to be retained in the Golgi. Indeed, retention may be developmentally regulated so that under certain conditions Cx40.8 is excluded from gap junctions, while under different conditions Cx40.8 is permitted to hetero-oligomerize with Cx43. Results from these aims will provide novel insights into the underlying mechanism of how Cx43 missense alleles cause ODDD disease phenotypes. In humans, missense alleles in CX43 are associated with oculodentodigital dysplasia. This disease is transmitted as autosomal dominant, indicating that the interaction of both wild-type and missense forms of the proteins contributes to the disease-related phenotypes. RELEVANCE: This application seeks to evaluate the interactions of zebrafish cx43 and a zebrafish cx43-like protein, cx40.8. The goal is to reveal if the co-expression of closely related connexin proteins can modify gap junctional properties in vitro, and cx43-related phenotypes in vivo.